Acanthamoeba profilin-1 is a 125 residue protein that binds to both actin and to the head groups of poly(phosphoinositide)s and may regulate both actin assembly and the phosphoinositide signaling pathway. In order to further the understanding of the activity of profilin at the molecular level we have determined its three dimensional structure using multi- dimensional heteronuclear NMR spectroscopy. A crystal structure of the protein has not yet been determined. The central feature of the profilin solution structure is a five stranded antiparallel beta sheet flanked by N- and C-terminal helices on one face, and by a third helix and a two stranded antiparallel beta sheet on the other face. Cross linking experiments and the location of conserved residues in profilins in different phyla suggest that actin binding occurs on the face of the protein occupied by the terminal helices. The other face of the molecule contains residues that may be important in determining the difference in polyphosphoinositide binding of profilin isoforms, suggesting that actin and lipid binding occur on adjacent faces of the protein. The significance of the project is that the profilin structure obtained provides the first information about the interactions of profilins with actin and lipids. These interactions are thought to link processes that regulate transmembrane signaling with formation of the cytoskeleton.